The Role of Zuo Jin Wan in Modulating the Tumor Microenvironment of Colorectal Cancer.

INTRODUCTION: Traditional Chinese medicine (TCM) can modulate the immune function of tumor patients in various ways. Zuojin Wan (ZJW, a 6:1 ratio of Huanglian and Wuzhuyu) can modulate the microenvironment of ulcerative colitis, but its role in regulating the CRC microenvironment remains unclear. Exploring the role of ZJW in CRC immunomodulation may improve the antitumor effect of existing immunotherapeutic strategies. MATERIAL AND METHODS: The active compounds of each herb in ZJW were obtained from the HIT2.0 database with literature evidence. Single-cell RNA sequencing data of CRC were obtained from published studies (PMID: 32451460, 32103181, and 32561858). Pathway enrichment was analyzed using the reactome database, and intergenic correlation analysis was performed using the corrplot R software package. ZJW-regulated gene expression was verified by RT-qPCR. RESULTS: Huanglian and Wuzhu contain 19 and 4 compounds, respectively. Huang Lian targets 146 proteins, and Wu Zhu Yu targets 28 proteins based on evidence from the literature. ZJW regulates a range of biological processes associated with immune function, including cytokine signaling and Toll-Like Receptor 4 (TLR4) cascade. ZJW regulates malignant CRC cells, immune cells (including T-cells, B-cells, mast cells, NK/NKT cells, and myeloid cells), and other non-immune cells (including endothelial cells, enteric glial cells, and pericytes). We confirmed that ZJW significantly downregulated the expression of TIMP1 and MTDH. CONCLUSIONS: ZJW regulates a range of cells in the CRC microenvironment, including malignant CRC, immune cells, and stromal cells. In CRC cell lines, downregulation of TIMP1 and MTDH by ZJW may play an important role in the immunomodulation in CRC.

Research on the molecular mechanism of Zuo Jin Wan combined with cetuximab inducing ferroptosis in KRAS mutant colorectal cancer cells / 中国临床药理学与治疗学

AIM: To investigate the mechanism and reversal effect of Zuo Jin Wan (ZJW) on cetuximab (CET) resistance in KRAS mutant colorectal cancer cell. METHODS: The mutation status of KRAS gene in SW620, Lovo, HCT116, HT29 and Caco2 cells were detected by Sanger sequencing. CCK-8 assay was used to detect the effects of ZJW, CET, ZJW combined with CET and CET, ZJW in combination with other cell death inhibitors on the survival rate of the above cells, and to observe the reversal effects of ZJW on CET-treated KRAS mutant cells (SW620, Lovo and HCT116). Flow cytometry, colorimetric method, and Fe

Integrated microbiome-metabolomics analysis reveals the potential therapeutic mechanism of Zuo-Jin-Wan in ulcerative colitis.

BACKGROUND: Dysregulation in gut microbiota and host cometabolome contributes to the complicated pathology of ulcerative colitis (UC), while Zuo-Jin-Wan (ZJW), a traditional Chinese medicine has shown therapeutic effects against UC with its underlying mechanism remains elusive. PURPOSE: This study utilized an integrated analysis combining gut microbiome and host cometabolism to disclose the potential therapeutic mechanism of ZJW on dextran sulfate sodium (DSS)-induced UC in rats. METHODS: We first evaluated the therapeutic effects of ZJW treatment in DSS-induced rat model. 16S rRNA sequencing, 1H NMR spectroscopy-based metabolomics and Spearman correlation analysis were conducted to explore the potential therapeutic mechanism during the treatment. RESULTS: Our results showed that UC symptoms in ZJW rats were significantly attenuated. Marked decline in microbial diversity in ZJW group was accompanied by its correspondent function adjustment. Specific enrichment of genus Bacteroides, Sutterella, Akkermansia and Roseburia along with the major varying amino acid metabolism and lipid metabolism were observed meantime. Metabolic data further corroborated that ZJW-related metabolic changes were basically gathered in amino acid metabolism, carbohydrate/energy metabolism and lipid metabolism. Of note, some biochemical parameters were deeply implicated with the discriminative microbial genera and metabolites involved in tricarboxylic acid (TCA) cycle and amino acid metabolism, indicating the microbiome-metabolome association in gut microbiota-metabolite-phenotype axis during UC treatment of ZJW. CONCLUSION: For the first time, integrated microbiome-metabolome analysis depicted that ZJW could alleviate DSS-induced UC in rats via a crosstalk between gut microbiota and host cometabolites.

Study on molecular mechanism of Zuojin Pills for gastroesophageal reflux disease based on network pharmacology / 国际中医中药杂志

Objective:To analyze the possible mechanism of Zuojin Pills on gastroesophageal reflux disease based on network pharmacology. Methods:By searching for the active constituent and protein targets of Zuojin Pills in TCMSP database,the protein names were converted into gene names in Uniprot database. Cytoscape 3.7.1 was used to draw the active constituent-target-medicine network diagram of Zuojin Pills and analyze the topological parameters. Then find the target of gastroesophageal reflux disease through OMIM,GeneCards,DRUGBANK database, find the intersection target of medicine and disease, perform PPI network analysis on the intersection target in STRING 11.0, and use the Metascape database to enrich the intersection target for further analysis. Cytoscape 3.7.1 was used to draw a network diagram of the active constituent- target-pathway of the medicine and to conduct a topology parameter analysis. Results:The main active constituent of Zuojin Pills in the treatment of gastroesophageal reflux disease are quercetin, Evodiamine, R-tetrahydroberberine, 1-methyl-2-nonyl-4-quinolone, berberine, etc. Targets include PTGS2, NOS3, MAPK1, EGFR, TNF, IL6, ERBB2, VEGFA, EGF, IL1B, etc., and these processes are mainly completed through inflammatory response, cancer, cell proliferation and apoptosis, cell connection, etc. Conclusions:The treatment of gastroesophageal reflux disease with Zuojin Pills is a complex process with multiple constituent, multiple targets, and multiple pathways. It is hoped that it which could provide reference for the future research on its mechanism of action.

Effect of Zuojin Pill on HIF-1α expression and apoptosis of thyroid cells in rats with autoimmune thyroiditis / 国际中医中药杂志

Objective:To investigate the effect of Zuojin Pill on the expression of hypoxia inducible factor-1α (HIF-1α) and apoptosis of thyroid cells in rats with autoimmune thyroiditis (AIT). Methods:Ninetysix rats were divided into control group, model group, standarlized protocol and low, medium and high dose Zuojin Pill groups. Except the control group, the rats in other groups were produced as AIT model. After successful modeling, rats in low-, medium-, and high-dose groups were intragastrically administered with 0.63, 1.26, and 2.52 g/kg Zuojin Pill respectively, standarlized protocol was given 6.25 mg/kg Tripterygium wilfordii polyglycoside, and the control group and model group were given the same amount of normal saline. The levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TGAb) were detected by enzyme-linked immunosorbent assay (ELISA); the activity of SOD and the content of MDA in thyroid tissue were detected by kit; the pathological changes of thyroid tissue were observed by HE staining; the apoptosis of thyroid cells was detected by TUNEL; the expression of HIF-1α, B-cell lymphoma-2 (Bcl-2), Bcl-2-related X protein (Bax) and Cleaved caspase-3 in thyroid tissues was detected by Western blot. Results:Compared with the model group, the content of TNF-α, TPOAb and TGAb in serum of rats in the low, medium and high dose Zuojin Pill groups significantly decreased, the content of IL-10 in serum significantly increased ( P<0.05). The SOD activity significantly increased, and the MDA content significantly decreased in the low, medium and high dose Zuojin Pill groups ( P<0.05). The apoptosis rate significantly decreased in the low, medium and high dose Zuojin Pill groups ( P<0.05). The expression of HIF-1α (0.48 ± 0.05, 0.63 ± 0.06, 0.86 ± 0.06 vs. 0.33 ± 0.03), Bcl-2 (0.48 ± 0.04, 0.59 ± 0.05, 0.68 ± 0.04 vs. 0.37 ± 0.04) significantly increased, the expression of Bax (0.67 ± 0.05, 0.53 ± 0.05, 0.40 ± 0.05 vs. 0.80 ± 0.06), Cleaved caspase-3 (0.64 ± 0.06, 0.51 ± 0.03, 0.36 ± 0.03 vs. 0.77 ± 0.05) significantly decreased in the low, medium and high dose Zuojin Pill groups ( P<0.05). Conclusion:Zuojin Pill could enhance the expression of HIF-1α, regulate the secretion of inflammatory factors, reduce the level of oxidative stress and thyroid autoantibody, and inhibit the apoptosis of thyroid cells.

Extracts of Zuo Jin Wan, a traditional Chinese medicine, phenocopies 5-HTR1D antagonist in attenuating Wnt/ß-catenin signaling in colorectal cancer cells.

BACKGROUND: In vitro and in vivo studies have shown that Zuo Jin Wan (ZJW), a herbal formula of traditional Chinese medicine (TCM), possessed anticancer properties. However, the underlying mechanism for the action of ZJW remains unclear. Various subtypes of 5-Hydroxytryptamine receptor (5-HTR) have been shown to play a role in carcinogenesis and cancer metastasis. 5-HTR1D, among the subtypes, is highly expressed in colorectal cancer (CRC) cell lines and tissues. The present study aimed at investigating effect of ZJW extracts on the biological function of CRC cells, the expression of 5-HTR1D, and molecules of Wnt/ß-catenin signaling pathway. METHODS: In this study, the effect of ZJW extracts on 5-HTR1D expression and Wnt/ß-catenin signaling pathway were investigated and contrasted with GR127935 (GR), a known 5-HTR1D antagonist, using the CRC cell line SW403. The cells were respectively treated with GR127935 and different doses of ZJW extracts. Proliferation, apoptosis, migration, and invasion of SW403 cells were compared between ZJW and GR127935 treatments. The expression of 5-HTR1D and signaling molecules involved in the canonic Wnt/ß-catenin pathway were determined by Western blot analysis. RESULTS: After ZJW extracts treatment and GR127935 treatment, G1 arrest in cell cycle of SW403 was increased. Cell apoptosis was pronounced, and cell migration and invasion were suppressed. SW403 cells showed a dose-dependently decreased expression of 5-HTR1D, meanwhile, ß-catenin level was significantly decreased in nucleus of cells cultured with GR127935. Treatment of ZJW extracts dose-dependently resulted in decreased 5-HTR1D and a concomitant reduction in the Wnt/ß-catenin signal transduction, an effect indistinguishable from GR127935 treatment. CONCLUSION: The anticancer activity of ZJW extracts may be partially achieved through attenuation of the 5-HTR1D-Wnt/ß-catenin signaling pathway.

Exploration of anti-cancer effects and mechanisms of Zuo-Jin-Wan and its alkaloid components in vitro and in orthotopic HepG2 xenograft immunocompetent mice.

BACKGROUND: Zuo-Jin-Wan (ZJW), a two-herb formula consisting of Coptis chinensis (CC) and Evodia rutaecarpa (ER), is commonly used in traditional Chinese medicine for the treatment of cancers. However, the efficacies and mechanisms of ZJW and its alkaloid components on cancers are still unclear. METHODS: Here we investigated the anti-cancer effects and mechanisms of ZJW, CC, ER, berberine, and evodiamine in cells and in intrahepatic xenograft mice. RESULTS: Treatment of HepG2 cells with ZJW, CC, ER, berberine, and evodiamine significantly displayed cytotoxic effects in a dose- and time-dependent manner. Hierarchical cluster analysis of gene expression profiles showed that CC and ZJW shared a similar mechanism for the cytotoxic effects, suggesting that CC was the active ingredient of ZJW for anti-cancer activity. Network analysis further showed that c-myc was the likely key molecule involved in the regulation of ZJW-affected gene expression. A human hepatoma xenograft model was established by intrahepatic injection of HepG2 cells containing nuclear factor-κB-driven luciferase genes in immunocompetent mice. In vivo bioluminescence imaging showed that cells had been successfully transplanted in mouse liver. Oral administration of ZJW for 28 consecutive days led to a significant decrease in the accumulation of ascites, the ratio of tumor-to-liver, and the number of transplanted cells in livers. CONCLUSIONS: In conclusion, our findings suggested for the first time that ZJW significantly suppressed human cancer cell growth in orthotopic HepG2 xenograft-bearing immunocompetent mice. Moreover, c-myc might play a potent role in the cytotoxic mechanisms of ZJW, CC, ER, berberine, and evodiamine.